This invention relates to nucleic acid and amino acid sequences of a human ena/VASP-like protein splice variant and to the use of these sequences in the diagnosis, treatment, and prevention of reproductive, immunological, vesicle trafficking, nervous system, developmental, and neoplastic disorders.
The control of cell morphology and motility requires the coupling of external stimuli to processes that alter the cytoskeletal architecture. The mechanical forces that drive morphological change and migration arise initially from the microfilament-based cytoskeleton. A large body of evidence links various signal transduction pathways to the formation of cellular outgrowths. The migration of neuronal growth cones is a well-studied mechanism for the actin-driven formation of membrane protrusions. In one example, the processes of axonal outgrowth are mediated by the Drosophila homolog of the c-Abl tyrosine is kinase (Abl) and the product of the Disabled gene (Dab). Homozygous mutants of Abl and Dab make few or no proper axonal connections. The defects caused by loss of Abl and Dab in Drosophila are ameliorated by mutations in the Enabled (Ena) gene. Ena protein is tyrosine phosphorylated and has a proline-rich core which binds to the SH3 domains of Abl protein and Src protein in vitro. The murine homolog of Ena (Mena) and ena/VASP-like protein have recently been described and are members of a family of related molecules that include vasoactive-stimulated phosphoprotein (VASP). These proteins share three distinct regions of similarity: an amino-terminal 115 amino acids (EVH1 domain); a proline-rich core; and a carboxy-terminal 226 amino acids (EVH2 domain). Mena has phosphotyrosine and phosphoserine moieties and binds Abl and Src SH3 domains. (Gertler, F. B. et al. (1996) Cell 87:227-239.)
Human platelet activation is inhibited by agents such as prostaglandins and nitric oxide donors, which elevate intracellular cAMP or cGMP levels. Activation of platelets is asociated with increased formation of intracellular F-actin. VASP is an abundant in vivo substrate for cyclic nucleotide-dependant protein kinases in platelets. VASP is a ligand for profilin, an actin-monomer binding protein that can stimulate the formation of F-actin. VASP is organized into three distinct domains. A central proline-rich domain contains a GPPPPP motif as a single copy and as a 3-fold tandem repeat, as well as three conserved phosphorylation sites for cyclic nucleotide-dependent protein kinases. A C-terminal domain contains a repetitive mixed-charge cluster which is predicted to form an alpha-helix. VASP expression in transiently transfected BHK21 cells was predominantly detected at stress fibers, at focal adhesions, and in F-actin-containing cell surface protrusions. In contrast, truncated VASP lacking the C-terminal domain was no longer concentrated at focal adhesions. These data indicate that the C-terminal domain is required for anchoring VASP at focal adhesion sites, while the central domain may mediate VASP interaction with profilin. (Ermekova, K. S. et al. (1997) J. Biol. Chem. 272:32869-32877.)
In comparison, Mena binds FE65, a neuronal protein which binds to the cytoplasmic portion of the xcex2-amyloid precursor protein (xcex2-APP). xcex2-APP is a precursor to xcex2-amyloid peptide, the major constituent of the extracellular plaques present in brain tissue from Alzheimer disease patients. Both VASP and Mena bind their respective adapter proteins (profilin or FE65) via distinct proline-rich regions and thus regulate adapter interaction(s) with other molecules. (Ermekova, K. S. et al, supra.) Proline-rich domains and proline clusters have been identified in many proteins, particularly those that are associated with synaptic vesicles and other secretory organelles. These domains and clusters act as protein-protein interaction modules. (Linial, M. (1994) Neuroreport. 5:2009-2015.) Two members of ena/VASP-like proteins have been recently isolated from mouse and rat and share 98.5% sequence identity. (Gertler, supra; and Ohta, S. et al. (1997) Biochem. Biophys. Res. Comm. 237:307-312.)
The discovery of a new human ena/VASP-like protein splice variant and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, and prevention of reproductive, immunological, vesicle trafficking, nervous system, developmental, and neoplastic disorders.
The invention is based on the discovery of a new human ena/VASP-like protein splice variant (EVL1), the polynucleotides encoding EVL1, and the use of these compositions for the diagnosis, treatment, or prevention of reproductive, immunological, vesicle trafficking, nervous system, developmental, and neoplastic disorders.
The invention features a substantially purified polypeptide, comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention further provides a substantially purified variant having at least 90% amino acid identity to the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also includes an isolated and purified polynucleotide variant having at least 90% polynucleotide identity to the polynucleotide encoding the polypeptide consisting of the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
Additionally, the invention provides a composition comprising a polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention further provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as an isolated and purified polynucleotide which is complementary to the polynucleotide encoding the polypeptide consisting of the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides an isolated and purified polynucleotide comprising a sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2, and an isolated and purified polynucleotide variant having at least 90% polynucleotide identity to the polynucleotide comprising the sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2. The invention also provides an isolated and purified polynucleotide which is complementary to the polynucleotide comprising the sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2.
The invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. In another aspect, the expression vector is contained within a host cell.
The invention also provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide encoding the polypeptide under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition consisting of a substantially purified polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a polypeptide consisting of the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as a purified agonist and a purified antagonist of the polypeptide.
The invention also provides a method for treating or preventing a reproductive disorder, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising substantially purified polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides a method for treating or preventing an immunological disorder, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising substantially purified polypeptide comprising the amino sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides a method for treating or preventing a vesicle trafficking disorder, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising substantially purified polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides a method for treating or preventing a nervous system disorder, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising substantially purified polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides a method for treating or preventing a developmental disorder, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising substantially purified polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides a method for treating or preventing a neoplastic disorder, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides a method for detecting a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in a biological sample containing nucleic acids, the method comprising the steps of: (a) hybridizing the complement of the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 to at least one of the nucleic acids of the biological sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide encoding the polypeptide in the biological sample. In one aspect, the nucleic acids of the biological sample are amplified by the polymerase chain reaction prior to the hybridizing step.